Sepsis is a syndrome of systemic toxicity caused by the presence of bacteria and/or bacterial products in the blood stream..sup.1,2 This condition, often caused by bacteria which are part of the body's normal flora, occurs frequently when the body's defense mechanisms are compromised or overwhelmed, as in patients treated with immunosuppressants, corticosteroids, or radiotherapy, in infants with systemic meningococcal infections, post-surgical patients, patients with chronic liver disease, or with severe burns..sup.3 Increasingly, this condition is also precipitated in individuals treated with antibiotics for serious systemic infections. In these latter situations, antibiotics, while killing the bacteria, also cause the release into the bloodstream of a variety of products from the disintegrating bacteria..sup.4-7
It is now well recognized by researchers and by clinicians that endotoxins, or lipopolysaccharides (LPS)--structural components of the outer membranes of Gram-negative bacteria.sup.8,9 --play a pivotal role in the causation of the sepsis syndrome..sup.10,11 Lipopolysaccharides, released from bacteria, either by the body's natural defense systems, or by antibiotics, are recognized by a variety of cell types in the body, important among which is the monocyte/macrophage,.sup.12 a subset of the white blood cells. When these cells sense the presence of lipopolysaccharide, they respond by producing numerous inflammatory mediators, including tumor necrosis factor-alpha (TNF-.alpha.), interleukin-1 beta (IL-1.beta.),.sup.13-19 interleukin-6,.sup.20-22 interleukin-8, interferon,.sup.23 prostaglandins,.sup.24-27 leukotrienes,.sup.28,29 platelet activating factor,.sup.30-32 and procoagulant tissue factor..sup.33,34 Other cells, such as the endothelium, produce nitric oxide..sup.18,35-39 The production of these mediators, under normal circumstances, is precisely regulated, and serves to orchestrate the body's defense mechanisms.
However, the unregulated overproduction of these substances.sup.40 lead to the clinical syndrome termed "septic shock"..sup.1,2 This syndrome is characterized by fever, hypotension, coagulopathy, hemodynamic derangement, tissue hypoperfusion, and multiple organ failure, which frequently culminates in death of the patient..sup.41-46 It is estimated that more than 300,000 cases of septic shock occur each year in the United States..sup.47
The sepsis syndrome, however, is by no means an exclusive sequel of Gram-negative infections; about half of the mortality attributable to septic shock is associated with serious systemic Gram-positive organisms.sup.47-49 which is clinically indistinguishable from that due to Gram-negative bacteria. Experimental data exist in the scientific literature which demonstrate that Gram-positive organisms can also induce a shock-like state in animal models.sup.50-54 mimicking in most respects, shock induced by LPS. LPS-induced.sup.55 as well as Gram-positive bacteria-induced systemic inflammatory responses.sup.51 are preventable by anti-TNF-.alpha.antibody. Gram-positive bacterial cell-walls,.sup.50,56 and organism-free supernatants.sup.57-59 obtained from Gram-positive bacterial cultures stimulate cytokine production from mononuclear cells. These observations, collectively, suggest that a final common pathway, involving proinflammatory cytokine overproduction,.sup.60-62 may underlie the pathogenesis of septic shock, irrespective of the nature of the causative organism.
However, unlike Gram-negative sepsis, in which lipopolysaccharide is the primary molecule which initiates the systemic inflammatory responses, it is as yet unclear which component (or components) of the Gram-positive bacterium triggers the cytokine reponse.sup.63 which ultimately overwhelms the normal homeostatic mechanisms of the body. Although increasing attention has focused on the cytokine-inducing properties of lipoteichoic acids (LTA),.sup.64,65 structural components of the Gram-positive cell wall,.sup.66 recent evidence, independently obtained from two different research groups, would appear to suggest that a minor high molecular weight glycolipid species which may or may not be chemically related to LTA.sup.67-69 is the dominant cytokine-inducing molecule. The biological activity of this component, like LPS, is substantially enhanced in the presence of CD14, an LPS receptor, and is blocked by anti-CD14 antibody; furthermore, this component competitively inhibits LPS binding to cell surfaces..sup.68 It is reasonable, therefore, to hypothesize from these exploratory studies, that there may be present in Gram-positive bacteria, molecules of relatively low abundance which share some general physicochemical properties with that of LPS.
The therapy of septic shock remains, to date, primarily supportive, consisting of antibiotics to treat the underlying infection, as well as hemodynamic and respiratory support. Specific modalities of treating septic shock aimed at controlling those pathophysiological mechanisms that lead to the systemic inflammatory response which ultimately manifests in shock are, unfortunately, as yet unavailable.sup.12,70,71 although clinical trials involving a variety of experimental approaches are currently in progress. With regard to the specific clinical problem of Gram-negative sepsis, one possible approach would be to target lipopolysaccharide itself by the use of an agent that would bind to and sequester this potent microbial product, thereby preventing its recognition by the monocyte/macrophage and other effector cells. This approach of "proximal intervention" would, in many respects, appear to be preferable to others directed at later events (e.g. therapeutic targeting of the inflammatory mediators), for once the monocyte/macrophage cell is activated, the cellular response is so diverse that a single pharmacological agent would be unlikely to modulate the effects of all the mediators produced. This approach of sequestering lipopolysaccharide, historically, has been addressed by the use of either polyclonal or monoclonal antibodies raised against the structurally conserved regions of lipopolysaccharide (so that the antibody would be cross-reactive against several lipopolysaccharide species from diverse Gram-negative bacteria)..sup.72-81 However, clinical studies with polyclonal antibodies.sup.82,83 have been difficult to interpret unequivocally.sup.84 in spite of the fact that statistically significant levels of protection were reported by the authors. Numerous clinical trials.sup.85-90 designed to test the therapeutic efficacy of monoclonal antibodies have failed to establish that the use of such antibodies are of clinical value. One possible reason could be that the region on the lipopolysaccharide molecule recognized by the antibodies is "cryptic" or hidden, at the molecular level, by other regions of the toxin molecule.
Several LPS-binding proteins of non-immunologic origin which are known to bind and neutralize the effects of endotoxin are currently being evaluated as candidate therapeutic agents. An endotoxin-binding protein.sup.91,92 obtained from the Horseshoe crab Limulus polyphemus (U.S. Pat. Nos. 5,627,266; 5,614,369) and a protein found in neutrophil granules, called Bactericidal/Permeability-increasing Protein (BPI) 93-103 (U.S. Pat. Nos. 5,652,332; 5,646,114; 5,643,570; 5,639,727; 5,523,2885,494,896; 5,447,913; 5,420,019; 5,348,942; 5,348,942) have been patented for potential application in the treatment of septic shock. These proteins bind to lipopolysaccharide and neutralize its toxicity. Yet more recently, another protein belonging to a family of phospholipid-binding proteins called Annexins.sup.104-108 has been patented (U.S. Pat. No. 5,658,877). This protein, presumably, also binds to LPS and inhibits its toxicity. Unfortunately, the production of these proteins for widespread use as therapeutic agents is likely to prove costly and will potentially impact significantly upon health care costs in treating this disease.
The toxic center of the lipopolysaccharide molecule is a glycolipid called lipid A, whose structure is shown diagrammatically in FIG. 1. Chemically, lipid A consists of a .beta.-(1,6)-linked bis-glucosamine backbone with amide-and ester-linked fatty acids, and two phosphate groups on the backbone at the 1 and 4' positions..sup.9,109-112 The structure of lipid A is highly conserved and therefore very similar among Gram-negative bacteria. Because it is the toxic center of the endotoxin molecule, it therefore presents a logical molecular target for compounds designed to bind lipopolysaccharide.
The anionic and amphiphilic nature of lipid A.sup.109,110,112 enables it to bind to numerous substances which are positively charged and also possess amphipathic character. However, the binding of such molecules to lipopolysaccharide alone does not neutralize LPS toxicity, which requires that certain molecular characteristics be mandatorily present if adequate neutralization of endotoxicity is to occur. We have, over the last several years, characterized the interactions of lipopolysaccharide with a number of classes of molecules including proteins,.sup.113,114 peptides,.sup.119-122 pharmaceutical compounds,.sup.123,124 and other synthetic polycationic amphiphiles..sup.125,126 We discovered, during these studies, that molecules with multiple protonatable positive charges which are so disposed that the distance between the charges are approximately equal to the theoretical distance between the two negative charges present on the two phosphate groups of the lipid A molecule enable the binding of such molecules to lipid A, and also to lipopolysaccharide, the parent molecule..sup.123,124 We have further determined that the presence of appropriately positioned hydrophobic groups enhances and stabilizes the binding of such molecules..sup.124,125
As discussed above, there is a sufficient body of knowledge in the scientific literature to render tenable the hypothesis that, because molecules that resemble LPS, or of its lipid A moiety, in gross physicochemical terms may exist in the Gram-positive organism, some cationic amphiphiles may also sequester these as yet uncharacterized cytokine-inducing molecules and inhibit the effects of Gram-positive organisms.
The present discovery reported in this document describes our studies designed firstly to demonstrate the use of such cationic amphiphilic molecules in binding lipopolysaccharide, and in inhibiting its toxicity in vitro and in vivo; secondly we show that such cationic amphiphilic molecules also inhibit the deleterious effects caused by Gram-positive organisms in an animal model. We do not as yet understand the basis or the mechanism by which such cationic amphiphilic molecules afford protection against Gram-positive bacteria, and we presume that it is a consequence of the binding and subsequent neutralization of one or more species of molecule(s) present in Gram-positive organism which bear some similarities to LPS, or of its lipid A moiety, in terms of general physicochemical properties. Representative examples of the cationic amphiphilic molecules employed in these studies (shown in FIG. 2) include 1,3-di-oleoyloxy-2-(6-carboxyspermyl)-propylamide (available commercially as "DOSPER" from Boehringer-Mannheim Corporation, Indianapolis, Ind.), 2,3-dioleoyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiu m trifluoroacetate ("DOSPA"; available as LipofectAMINE.TM. from Life Technologies, Gaithersburg, Md.), dioctadecylamidoglycylspermine ("DOGS"; available as Transfectam.TM. from Promega Corporation, Madison, Wis.).
These compounds were originally developed.sup.127-133 and are currently being marketed as agents that will facilitate the transfection (transport) of DNA into recipient cells. A list of U.S. patents related to the development and use of these molecules, that have been filed to date is provided in Table 1.
TABLE 1 __________________________________________________________________________ Partial list of U.S. Patents relating to the development and applications of lipopolyamine compounds CATEGORY U.S. Pat. No. INVENTORS TITLE __________________________________________________________________________ Lipofect- 5,627,159 Shih P J et al Enhancement of lipid cationic transfections . . . AMINE .TM. 5,578,475 Jessee J A Composition and methods for transfecting . . . Transfectam .TM. 5,650,096 Harris D J et al Cationic amphiphiles for intracellular delivery . . . 5,635,487 Wolff J A et al Amphipathic, micellar delivery systems . . . 5,616,745 Behr J P et al Lipopolyamines, their preparation and their use 5,476,962 Behr J P et al New lipopolyamines, their preparation and their use 5,171,678 Behr J P et al Lipopolyamines, their preparation and use DOSPER 5,661,018 Ashley G W et al Cationic phospholipids for transfection 5,651,981 Ashley, G W et al Cationic phospholipids for transfection 5,650,096 Harris D J et al Cationic amphiphiles for intracellular delivery . . . 5,283,185 Epand R M et al Method for delivering nucleic acids into cells OTHER 5,521,291 Curiel D T et al Conjugates for introducing nucleic acid . . . 5,614,503 Chaudhary N et al Amphipathic nucleic acid transporter 5,635,380 Naftilan A J et al Enhancement of nucleic acid transfer . . . 5,342,945 Bergeron R J et al Anti-neoplastic, anti-viral, or . . . 5,527,928 Nantz M H et al Cationic transport reagents 5,459,127 Felgner, P L et al Cationic lipids for intracellular delivery . . . 5,264,618 Felgner P L et al Cationic lipids for intracellular delivery . . . __________________________________________________________________________
Nonetheless, there remains a need in the art for a demonstration of the use of such molecules in binding lipopolysaccharide and in inhibiting its toxicity.